I work with large proteins and try to transfer my western blot studies for his reason ı using 6% gel. In a semi-dry transfer system, my all proteins are transferred in 25V 1.5amper 9 minutes ı check my jel with commasie blue stain. Then bloking at 5% BSA TBST 1 hour at room temperature for 1 hour, wash three times, 1 hour at room temperature in 1:1000 primary antibody in 1% TBST BSA and wash three times, and finally, 1% TBST BSA 1 hour at room temperature 1:5000 secondary antibody. But the membrane seems very dark and dirty. After mild stripping of the membranes, I can only get imaging, but ı think that after mild stripping ı lost my protein because the bands seem very reduced.